A survey on the incidence and magnitude of intestinal helminthiasis in broiler breeders originating from the southeastern United States 1

2013 Poultry Science Association, Inc. A survey on the incidence and magnitude of intestinal helminthiasis in broiler breeders originating from the southeastern United States 1 T. Yazwinski,* 2 C. Tucker,* E. Wray,* L. Jones,* Z. Johnson,* S. Steinlage, and J. Bridges * Department of Animal Science, University of Arkansas, Fayetteville 72701; and Elanco Animal Health, Greenfield, IN 46140 Primary Audience: Breeder Flock Managers, Live Production Managers, Veterinarians, Nutritionists SUMMARY A survey was conducted to determine the incidence and magnitudes of parasitic helminth populations in broiler breeders. To that end, intact intestinal tracts were submitted by personnel from 10 poultry companies located in the southeastern United States. A total of 281 intestines were submitted, with 5 to 6 intestines being submitted from each of 47 breeder barns representing the 10 poultry companies. The birds selected for sacrifice were obtained at random from each barn, and ranged in age from approximately 30 to 49 wk at posting. Intestines were extracted on site, placed individually in plastic bags, chilled, and mailed overnight to the University of Arkansas for parasite collection, identification, and quantification. All intestines were identified with company, farm, bird age, and grower ranking. The majority of intestines were submitted with information relative to anthelmintic treatments given previously to the birds in the pullet house as well as specifics concerning bedding (number of flocks on current bedding). Of the 281 intestines submitted in the survey, only 3 were found to be void of helminth parasites, resulting in an overall infection incidence of 98.9%. Helminths isolated and identified from the intestinal tracts (and overall incidences) were Heterakis gallinarum (96%), Capillaria obsignata (75%), Ascaridia galli (63%), and Raillietina cesticillus (14%). Helminth counts for individual birds ranged from zero to a maximum of 3,240, 1,280, 940, and 445 for H. gallinarum, C. obsignata, A. galli, and R. cesticillus, respectively. Helminth levels varied significantly by company, but not significantly by grower ranking, although a direct correlation was evident between each parasite population and grower ranking (lower producer rankings were associated with higher helminth burdens). No correlation was detected between the helminth levels as seen in the surveyed birds and prior anthelmintic usage when the birds were in pullet production an illustration of the refractory or compensatory nature of helminths and the inability of current treatments to control helminthiasis in breeders. Key words: chicken, helminth infection, incidence, magnitude 2013 J. Appl. Poult. Res. 22 :942 947 http://dx.doi.org/10.3382/japr.2013-00776 1 This research was funded in part by Elanco Animal Health, 2500 Innovation Way, Greenfield, IN 46140. 2 Corresponding author: yazwinsk@uark.edu

Yazwinski et al.: INTESTINAL HELMINTHIASIS 943 DESCRIPTION OF PROBLEM Commercial poultry in the United States are parasitized by many organisms, including internal and external parasites, and metazoan and protozoan parasites. Infections by protozoan parasites (e.g., Eimeria and Histomonas species) are usually under surveillance, as these infections are capable of becoming acutely manifest with significant economic and bird losses. External parasites (e.g., mites, lice) are usually visible to the producer either directly as organisms or indirectly via their lesions and altered bird activity, with infestations dealt with as warranted. In contrast to the above, infections by helminth parasites are generally subtle and nonclinical and manifest only indirectly by reduced appetite and feed efficiency. Additionally, parasitic helminth populations in poultry more than likely have the same adverse effects on immune competence of the host as has been documented in other animals and in humans [1], a consequence of helminthiasis that would adversely affect the immunological response of birds to other infections and vaccines. Helminth infections of poultry convey significant negative effects on bird production, but given the fact that helminth infections are subclinical, controls exercised by producers are extremely varied and generally not monitored for effectiveness. Generally, helminth infections are viewed by personnel in the poultry industry as significant but their presence is accepted as inevitable. The current survey was conducted to determine the level of helminthiasis in broiler breeders, a segment of the poultry industry that has not been previously evaluated for helminth presence, but one that is vital to broiler production. With the results of this survey, a good estimate of helminth incidence and magnitude can be derived for broiler breeders in the southeastern United States. MATERIALS AND METHODS Sample Acquisition Intestines (intact from the gizzard to the vent) were obtained from randomly gathered birds (30 to 49 wk of age) at broiler breeder houses at the sampling rate of 5 to 6 birds per house. Some of the sampled birds were male but most were female. In total, 47 houses representing 3 tiers of production efficiency at 10 poultry companies were sampled. The intestines were individually bagged by on-site personnel, and sent to the University of Arkansas (Fayetteville) along with an information collection form with various degrees of completion (e.g., prior anthelmintic usage, bird age, production efficiency ranking, litter specifics, recent or current disease state of the flock). Parasitology Immediately upon arrival at the parasitology laboratory, the intestines were processed for parasite collection, identification, and quantification according to descriptions and procedures published by the World Association for the Advancement of Veterinary Parasitology [2] and elsewhere [3]. Because all intestines were processed for parasite isolation 24 h following sacrifice, there was no need for overnight soaking of the tracts for parasite dissociation from the tissues before parasite collection. The intestines were opened lengthwise (pyloric sphincter to rectum, inclusive of the ceca), and all contents (with thorough rinsing) were washed over a number 200 sieve (aperture of 75 μm) and the residue viewed in its entirety for adult Ascaridia galli. Subsequent to the adult ascarid count, the entire residue from each tract was then subsampled (with subsamples ranging from 10 to 100% of total depending on amount of residue and number of parasites to be counted) for stereoscopic viewing and counting of the smaller helminth forms (cestode scolexes, immature stages of A. galli, and all stages of Heterakis gallinarum and Capillaria obsignata). Statistics Arithmetic means and standard deviations were generated for all helminth populations by standard calculations. Statistically significant mean separations were determined by repeated t-tests at the 5% level of confidence after ANO- VA of transformed data (log 10 [x + 1]) and the generation of a significant F statistic (P < 0.05) for the overall model. Only arithmetic means (with SD) are presented.

944 JAPR: Field Report Table 1. Levels of helminths determined for the 281 submitted intestines Species Range Arithmetic mean Incidence (% infected) Ascaridia galli 0 940 32.9 63 Heterakis gallinarum 0 3,240 285.4 96 Capillaria obsignata 0 1,680 142.8 75 Raillietina cesticillus 0 445 3.9 14 RESULTS AND DISCUSSION An overall summary of the helminth populations (incidences, means, and ranges by helminth) detected in this survey is given in Table 1. A ranking of all helminths identified in this survey, regardless of parameter (incidence, mean, or range) was H. gallinarum > C. obsignata > A. galli > Raillietina cesticillus. These helminths are also the most commonly found helminths of confined chickens in the southeastern United States. All helminths cited above have been shown to confer significant detriment to their parasitized host when they are established at the levels that were seen in this survey. Infections with H. gallinarum result in inflammation and thickening of the cecal mucosa and submucosa, along with varying degrees of nodule formation [4]. The primary significance of H. gallinarum, however, might well be its obligatory role in the reservoir and transmission of Histomonas meleagridis, the causative agent of histomoniasis ( black head disease) [5]. In the current survey, 55.2% of the cecal worms were at the adult stage, with the remaining 44.8% at a larval stage. Infections with C. obsignata result in a thickening of the small intestine wall and excessive mucus production, pathogenesis that leads to anorexia, diarrhea, reduced feed efficiency, and even mortality [6]. Given the filamentous morphology of C. obsignata, it is likely that the nematode entwines itself around villi, resulting in villus atrophy and rejection (thigmokinetic effect). No attempt was made to identify the isolated C. obsignata to development stage, as this would have proven impossible in most cases because Capillaria spp. are usually isolated in pieces (the result of washing contents on sieves). Of all the helminths quantified in this survey, A. galli has received the most research attention relative to pathogenesis, life cycle dynamics, susceptibility to chemotherapy, and so on. Additionally, A. galli is noted for an extensive histotropic nature during the development (and arrestment) of parasitic larvae [7]. Ascarid infections of chickens commonly result in anorexia, reduced feed efficiency, and weight loss, with mortality an occasional observation in younger birds due to mucosal inflammation or intestinal impaction [8]. Ascarid infections of chickens are primarily confined to younger birds [9], and the relatively high incidence of these parasites in the surveyed birds (63% infected) was unexpected. Only 8.5% of the isolated A. galli forms were at the adult stage, with the majority of the population at larval stages (L2, L3, and L4 were 30.8, 47.9 and 10.7% of the average total ascarid burdens, respectively), a reflection of pronounced immunity-based inhibition of the ascarid infections in poultry [10]. Raillietina cesticillus was the only cestode isolated in the survey, with 14% of the birds infected. Generally, infections were very light (mean scolex count of 3.9), although some infections were quite extensive (high single scolex count of 445). Tapeworm infections similar to those seen in this survey have been found to be responsible for reductions in bird weight and productivity [11]. Helminth levels by company are given in Table 2. Significant differences between companies were apparent for each parasite. Generally, company ranking by helminth arithmetic mean counts (and statistical separation) was roughly consistent across all parasites. For example, company 1 was in the most heavily parasitized grouping of companies (by means and statistical significance) for each of the 4 helminth species. Conversely, company 4 was in the least parasitized grouping for each helminth. Clearly, practices and conditions distinct to each company were critical in determining the magnitude of parasite populations, with conditions that opti-

Yazwinski et al.: INTESTINAL HELMINTHIASIS 945 Table 2. Helminth levels by company (arithmetic means and ranges) Species Ascaridia galli Heterakis gallinarum Capillaria obsignata Raillietina cesticillus Company N Mean Range Mean Range Mean Range Mean Range 1 18 71.8 a 7 286 703.4 ab 5 3,240 515.4 a 75 1,070 8.8 ab 0 150 2 36 32.4 bc 0 362 174.9 d 0 640 62.7 d 0 315 2.1 b 0 15 3 53 34.4 cd 0 940 254.5 ab 0 1,740 147.4 b 0 1,680 0 c 0 4 18 0.6 e 0 10 60.3 d 0 350 11.4 d 0 50 0.6 bc 0 5 5 54 38.9 b 0 180 283.2 ab 0 1,610 162.0 bc 0 1,280 1.1 b 0 20 6 18 13.8 bcd 0 56 155.3 bcd 0 550 92.5 c 0 415 0 c 0 7 18 59.9 a 0 178 418.1 a 20 1,560 106.7 c 0 455 41.9 a 0 445 8 12 0.2 e 0 2 194.6 bc 25 395 57.1 d 0 220 0.4 bc 0 5 9 18 39.8 a 0 145 184.4 bc 45 400 100.8 c 0 255 1.7 b 0 10 10 36 15.9 d 0 85 425.1 a 5 3,110 159.7 c 0 460 0.4 bc 0 10 a e Species-specific means with different superscripts are different with transformed data [Log 10 (x + 1)] (P < 0.05). mized the epidemiology of one helminth optimizing the others as well. A similar significant company effect in regard to parasite incidence and magnitude was seen earlier in a survey of helminths in broiler chickens [12]. Parasite levels by grower ranking (a production efficiency descriptive consistent for each company) are given in Table 3. No statistically significant differences were seen for the parasite means between the 3 tiers of grower ranking (transformed data; P < 0.05). However, for each nematode population, the tendency was for higher values as the grower ranking increased (higher nematode counts correlating with lower production efficiencies). The opposite tendency was seen for R. cesticillus (more efficient farms had higher tapeworm populations). Helminth levels in the breeder birds as a function of anthelmintic regimen used previously for the birds during their pullet production phase are given in Table 4. Information regarding prior anthelmintic usage was provided by submitting companies for 233 of the 281 submitted intestines. Pullet treatments could be roughly characterized into 5 regimens: repeated fenbendazole, repeated albendazole, piperazine treatment alternated with fenbendazole, piperazine treatment alternated with albendazole, and no treatment. No tendencies or significant mean separations were seen between treatment regimens for A. galli, C. obsignata, or R. cesticillus. A significant difference was observed between regimens (transformed data; P < 0.05) relative to H. gallinarum population means. This observation is extremely tentative due to the fact that all birds in the statistically significant low H. gallinarum group were from one company, thereby confounding company effect with that of treatment. Within the limitations of this survey, therefore, it does not appear that the anthelmintic regimens used by the companies during the pullet phase of breeder bird production result Table 3. Helminth levels by grower ranking Nematode Ascaridia galli Heterakis gallinarum Capillaria obsignata Ranking 1 N Mean Range Mean Range Mean Range 1 90 24.1 0 279 261.1 0 3,110 131.4 0 840 2 101 38.6 0 940 271.7 0 1,860 134.9 0 1,070 3 90 35.2 0 362 325.1 3 3,240 163.2 0 1,180 1 The higher the number, the lower the efficiency of production.

946 JAPR: Field Report Table 4. Helminth levels in the sampled breeder birds (by drug regimen used during pullet production) Arithmetic mean (SD) for total burden as Regimen 1 N Ascaridia galli Heterakis gallinarum Capillaria obsignata Raillietina cesticillus 1 54 44.1 (66.1) 393.9 a (691.1) 211.2 (283.1) 17.1 (64.9) 2 113 35.2 (97.8) 269.8 a (350.5) 148.4 (296.8) 0.4 (1.9) 3 18 22.6 (43.9) 84.3 b (137.9) 56.2 (54.4) 1.6 (4.1) 4 30 31.9 (40.3) 195.5 a (170.3) 90.0 (105.6) 2.0 (4.3) 5 18 39.8 (36.6) 184.4 a (117.2) 100.8 (90.4) 1.7 (3.0) a,b Species-specific means with different superscripts are different with transformed data [Log 10 (x + 1)] (P < 0.05). 1 1 = repeated fenbendazole treatments; 2 = repeated albendazole treatments; 3 = repeated piperazine and fenbendazole treatments; 4 = repeated piperazine and albendazole treatments; and 5 = no treatments. in altered helminth loads in the breeder birds. Actual efficacies of the anthelmintics used during the rearing of the study birds cannot be elucidated from these results, as many factors other than true anthelmintic efficacy undoubtedly contributed to the populations of the helminths quantified in the survey (e.g., bird challenge posttreatment, efficiency of drug deliveries, dosage rates). In early studies, however, piperazine was shown to confer little efficacy regardless of dose rate [13], and benzimidazoles (fenbendazole and albendazole) have been shown to elicit good levels of efficacy, which most recently appear to be on the decrease [14]. CONCLUSIONS AND APPLICATIONS 1. Helminth infections of broiler breeders appear to be the norm, with 98.6% of sampled birds harboring a combination of A. galli, H. gallinarum, C. obsignata, and R. cesticillus. In addition to extremely high prevalence, each helminth was found to infect the birds in high numbers (maximum numbers per bird of 3,240, 1,280, 940, and 445 for H. gallinarum, C. obsignata, A. galli, and R. cesticillus, respectively). The levels of helminthiasis observed in this survey coincide with diminished bird performance, as the helminth infections observed in this survey are higher than those cited in other research wherein birds were adversely affected. 2. Given the results of the current research, several follow-up investigations are indicated. First, a study designed to follow helminth levels from hatchery to breeder house is warranted wherein housing parameters, bird genetics, husbandry practices, and anthelmintic efficacies are monitored. Second, a performance study should be conducted so that the effect of normal worm burdens might be quantified relative to bird performance and economics. Third, anthelmintic efficacy studies should be performed so that efficacies might be documented for the products we have available. Finally, investigational new animal parasiticides should be screened for effectiveness in the chicken-helminth model. 3. Results from the current study indicate that helminth infections of broiler breeders are extremely common and of high magnitude, existing at levels of infection that would translate into production and economic losses in the field. Increased diligence should be exercised by the poultry industry to curb these parasitisms, a tremendously involved task given the number of factors in play (e.g., helminth species, drug efficacies, helminth persistence in the environment, complexities of husbandry and housing schemes, and so on). REFERENCES AND NOTES 1. Maizels, R. M., and M. Yazdanbakhsh. 2003. Immune regulation by helminth parasites: Cellular and molecular mechanisms. Nat. Rev. Immunol. 3:733 744. 2. Yazwinski, T. A., H. D. Chapman, T. J. Davis, R. B. Letonja, L. Pote, L. Maes, J. Vercruysse, and D. E. Jacobs.

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